Melanoma is a major medical problem with a rapidly increasing incidence and a growing lifetime risk. Treatment options, cure rates and survival decrease dramatically with disease progression. Despite concerted efforts, chemotherapy regimens have so far yielded disappointing results, related to intrinsic resistance. Apoptosis-based chemogene therapy (defined as drug-induced target gene upregulation/ activation) is emerging as a viable and promising alternative to conventional chemotherapy. However, the selection of the appropriate target is a major clinical challenge. We have recently cloned a novel heat shock protein, H11, that differs from other known family members in that it has cell type and stimulus specific pro- apoptotic activity upon overload. In melanoma, H11 is silenced by aberrant DNA methylation. Its forced expression by drug-induced de-methylation (viz. with Aza-C) triggers growth arrest/apoptosis, which are inhibited by H11 specific oligonucleotides (ODNs), suggesting that H11 is a promising target for chemogene therapy. Three specific Aims are proposed to test this hypothesis. Aim I will use real time methylation specific PCR and quantitative RT-PCR to examine the frequency of H11 silencing in melanoma and nevi tissues, normal melanocyte cultures and early passage freshly isolated melanoma cultures. Aim II will use de-methylation (Aza-C, with or without H11 antisense ODN and controls) and Dox treatment of melanoma cells stably transfected with Tetracycline regulated H11, to extend present findings for TAK1 function in Nil-induced growth arrest/apoptosis. Studies will confirm H11/TAK1 binding, identify binding sites, define the mechanism of H11-induced TAK1 activation, and verify the role of TAK1-mediated inhibition of beta- catenin. Additional early passage melanoma cultures (5-6/year) will be studied. Studies of normal melanocytes and keratinocytes will confirm the failure of Aza-C to cause H11 overload and growth arrest/ apoptosis, verify H11 sequestration through Hsp27 binding and the ability of H11 to potentiate Hsp27- mediated AKT activation/cell survival. Aim III will document that H11 overload kills melanoma xenografts and examine the ability of H11 overload to sensitize melanoma cells to cytotoxic drugs in culture and in vivo. The proposed studies will elucidate a novel paradigm for Hsp function in cancer cell fate determination, develop a much needed novel chemogene therapy for melanoma and identify targets for future H11-based therapies.